The invention relates to nucleic acids covalently coupled to electrodes via conductive oligomers. More particularly, the invention is directed to the site-selective modification of nucleic acids with electron transfer moieties and electrodes to produce a new class of biomaterials, and to methods of making and using them.
The detection of specific nucleic acids is an important tool for diagnostic medicine and molecular biology research. Gene probe assays currently play roles in identifying infectious organisms such as bacteria and viruses, in probing the expression of normal genes and identifying mutant genes such as oncogenes, in typing tissue for compatibility preceding tissue transplantation, in matching tissue or blood samples for forensic medicine, and for exploring homology among genes from different species.
Ideally, a gene probe assay should be sensitive, specific and easily automatable (for a review, see Nickerson, Current Opinion in Biotechnology 4:48-51 (1993)). The requirement for sensitivity (i.e. low detection limits) has been greatly alleviated by the development of the polymerase chain reaction (PCR) and other amplification technologies which allow researchers to amplify exponentially a specific nucleic acid sequence before analysis (for a review, see Abramson et al., Current Opinion in Biotechnology, 4:41-47 (1993)).
Specificity, in contrast, remains a problem in many currently available gene probe assays. The extent of molecular complementarity between probe and target defines the specificity of the interaction. Variations in the concentrations of probes, of targets and of salts in the hybridization medium, in the reaction temperature, and in the length of the probe may alter or influence the specificity of the probe/target interaction.
It may be possible under some limited circumstances to distinguish targets with perfect complementarity from targets with mismatches, although this is generally very difficult using traditional technology, since small variations in the reaction conditions will alter the hybridization. New experimental techniques for mismatch detection with standard probes include DNA ligation assays where single point mismatches prevent ligation and probe digestion assays in which mismatches create sites for probe cleavage.
Finally, the automation of gene probe assays remains an area in which current technologies are lacking. Such assays generally rely on the hybridization of a labelled probe to a target sequence followed by the separation of the unhybridized free probe. This separation is generally achieved by gel electrophoresis or solid phase capture and washing of the target DNA, and is generally quite difficult to automate easily.
The time consuming nature of these separation steps has led to two distinct avenues of development. One involves the development of high-speed, high-throughput automatable electrophoretic and other separation techniques. The other involves the development of non-separation homogeneous gene probe assays.
PCT application WO 95/15971 describes novel compositions comprising nucleic acids containing electron transfer moieties, including electrodes, which allow for novel detection methods of nucleic acid hybridization.
Accordingly, it is an object of the invention to provide for improved compositions of nucleic acids covalently attached to electrodes and at least one other electron transfer moiety.
In one aspect, the present invention provides methods for detecting the presence of a target sequence in a nucleic acid sample. The methods comprises applying a first input signal comprising an AC component and a non-zero DC component to a hybridization complex comprising at least a target sequence and a first probe single stranded nucleic acid. The hybridization complex is covalently attached to a first electron transfer moiety comprising an electrode, and a second electron transfer moiety. The presence of the hybridization complex is detected by receiving an output signal characteristic of electron transfer through said hybridization complex.
In an alternative aspect, the invention provides methods as above wherein a first input signal comprising an AC component at a first frequency and a non-zero DC component is applied to a hybridization complex, and then a second input signal comprising an AC component at least a second frequency and a non-zero DC component is applied, such that the presence of the hybridization complex can be detected.
In a further aspect of the invention, the methods utilize a first input signal comprising an AC component and a first non-zero DC component applied to a hybridization complex, followed by a second input signal comprising said AC component and at least a second non-zero DC component.
In an additional aspect, the invention provides methods that utilize a first input signal comprising an AC component at a first voltage amplitude, and a second input signal comprising said AC component at a second voltage amplitude applied to the hybridization complex.
In an additional aspect, the invention provides methods utilizing hybridization complex comprising a single stranded nucleic acid covalently attached to both a first electron transfer moiety comprising an electrode, and a second electron transfer moiety, and a target sequence hybridized to said single stranded nucleic acid.
In a further aspect, the hybridization complexes comprise a single stranded nucleic acid covalently attached via a conductive oligomer to a first electron transfer moiety comprising an electrode, and a target sequence hybridized to said single stranded nucleic acid, and a second electron transfer moiety.
In a further aspect, the invention provides apparatus for the detection of target nucleic acids in a test sample, comprising a test chamber comprising a first and a second measuring electrode, wherein the first measuring electrode comprises a covalently attached conductive oligomer covalently attached to a single stranded nucleic acid, and an AC/DC voltage source electrically connected to the test chamber.
In an additional aspect, the invention provides apparatus comprising a test chamber comprising a first and a second measuring electrode, wherein the first measuring electrode comprises a covalently attached single stranded nucleic acid comprising a covalently attached second electron transfer moiety, and an AC/DC voltage source electrically connected to the test chamber.